The long-term objective of the proposed studies is to develop intracellular immunization approaches for the treatment of AIDS. In the course of the previous funding period, we have developed a novel inhibition strategy utilizing TAR decoys to render CEM cells (a CD4+ human T-cell line) highly resistant to HIV replication. Towards the development of a peripheral T-cell targeted approach for the treatment of AIDS, I propose in this application to develop the conditions to transfer HIV resistance to short-term cultures of peripheral CD4+ T cells (PBL) obtained from human volunteers. In preliminary studies we have shown that retroviral-mediated transfer of TAR decoy templates into normal CD4+ T cells led to a reduction of HIV replication and also provided a measure of protection from the cytopathic effects of HIV. However, the extent of inhibition observed in PBL was less dramatic than that observed in previous studies using an established cell line (CEM). The specific aims of the proposed studies are: (1) to improve the effectiveness of the TAR decoy-mediated inhibition strategy, primarily by increasing the concentration of TAR decoy RNA expressed in the cell. This will be achieved by developing improved pol III-based systems derived from tRNA and U6 snRNA genes. (2) To develop the conditions for inhibition of HIV replication in cultured peripheral blood lymphocytes obtained from HIV seronegative volunteers. Conditions will be sought that maximize efficiency of retroviral transfer of TAR decoy templates while minimizing ex-vivo manipulation of normal peripheral blood lymphocytes.